TCA Cycle

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28 August 2006
TCA Cycle
Dr. Maurice Manning, Ph.D.


TCA Cycle Overview

  • Tricarboxyclic Acid Cycle
  • Takes Acetic Acid and 2 H2O to break that down into 2 CO2 and 4 H2
    • H2 is used to generate ATP
  • Fully oxidize acetic acid
  • Carbohydrates, proteins and lipids all feed in to make Acetyl-CoA
  • Takes place in the mitochondria
  • Requires oxygen to be the ultimate receptor of electrons from hydrogen
  • In total, 3 NADH+H+, 1 FADH2+ and 1 ATP is made per turn
    • 3 NADH+H+, 1 FADH2+ go on to oxidative phosphorylation
      • NADH+H+ generate 2.5 ATP
      • FADH2+ generates 1.5 ATP
  • In total, 8 reactions
    • Citrate Synthase
    • Aconitase
    • Isocitrate Dehydrogenase
    • Α-ketoglutarate Dehydrogenase
    • Succinyl-CoA Synthetase
    • Succinate Dehydrogenase
    • Fumarase
    • Malate Dehydrogenase
  • 10 intermediates
    • Acetyl CoA (C2)
    • Oxaloacetate (C4)
    • Citrate (C6)
    • Cis-Aconitate (C6)
    • Isocitrate (C6)
    • Α-Ketoglutarate (C5)
    • Succinyl-CoA (C4)
    • Succinate (C4)
    • Fumarate (C4)
    • Malate (C4)

TCA Cycle in Schematic

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TCA Cycle in Detail

Reaction 1


  • Acetyl CoA + Oxaloacetate --> Citrate
    • Uses 1 H2O and lose 1 CoA-SH
    • Catalyzed by citrate Synthase

Reaction 2

  • Citrate --> cis-Aconitate
    • H2O is lost
    • Catalyzed by Aconitase
    • Occurs as an equilibrium reaction
    • Citrate can be used to make fat
    • Citrate is a pro-chiral molecule
      • Although citrate is symmetrical, it reacts asymmetrically with aconitase
  • Cis-Aconitate --> isocitrate
    • H2O is reclaimed and the –OH is moved to the β-carbon
    • Catalyzed by Aconitase
    • Occurs in an equilibrium reaction

Reaction 3

  • Isocitrate --> α-ketoglutarate
    • First CO2 is lost, first NADH+H+ is generated
    • Catalyzed by Isocitrate dehydrogenase
    • This is the committed step of the TCA cycle
      • Decarboxylation and Dehydrogenation occur in this step
    • Strongly inhibited by the negative modulators NADH+H+ and ATP
    • Stimulated by ADP

Reaction 4

  • α-ketoglutarate --> Succinyl-CoA
    • Second CO2 is lost, second NADH+H+ is generated
    • Analogous to pyruvate dehydration reaction
      • Requires thyamaine, lipoic acid, co-enzyme A, FAD+, and NAD+
    • Catalyzed by α-ketoglutarate dehydrogenase
    • Succinyl-CoA is a high-energy thio-ester
    • Dehydrogenation step

Reaction 5

  • Succinyl-CoA --> Succinate
    • Phosphorylation of GDP to GTP by substrate-level phosphorylation
      • GTP later converted to ATP by nucleoside diphosphate kinase
    • Catalyzed by Succinyl CoA synthase
    • Succinyl-CoA is a precursor for heme
    • Odd-numbered fatty acids will oxidize to a C3 fatty acid which can become succinyl-CoA for further metabolism

Reaction 6

  • Succinate --> Fumarate
    • FADH2 is generated
      • Hydrogens removed in a trans fashion
    • Catalyzed by succinate dehydrogenase
    • Dehydrogenation step
    • This as an equilibrium step

Reaction 7

  • Fumarate --> Malate
    • H2O is lost
    • Catalyzed by fumarase

Reaction 8

  • Malate --> Oxaloacetate
    • The third NADH+H+ is generated
    • Catalyzed by malate dehydrogenase
    • Dehydrogenation step

Overall Reaction

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O --> 2 CO2 + 3 NADH+H+ + FADH2 + GTP

Acetyl CoA Formation

  • Formed from pyruvate from glucose or from fatty acids by β-oxidation
  • Pyruvate + NAD+ + CoA --> Acetyl-CoA + NADH+H+ + CO2
  • Catalyzed using Pyruvate Dehydrogenase Complex (PDC)
    • Involves thyamaine paraphosphate, lipoic acid, co-enzyme A, FAD+, and NAD+
  • Reaction is reversible in animal tissues
  • Obligatory step for the entry of all carbohydrates in the TCA cycle
  • Reaction requires multienzyme PDC of 3 different enzymes, 5 different co-enzymes, plus 2 enzymes which regulate PDH
    • Enzymes required are:
      • Pyryvate Dehydrogenase (regulatory enzyme)
      • Dihydrolipoyltransacetylase
      • Diydrolipoyl’ dehydrogenase
    • Five required co-enzymes are:
      • Thiamine paraphosphate
      • Lipoic Acid
      • Co-enzyme A
      • FAD
      • NAD+
  • Pyruvate + Co-Enzyme A --> Aceytl CoA + NADH+H+, CO2

Regulation of Pyruvate Dehydrogenase

  • Covalent Modification
    • Active PDH is unphosphorylated
      • Ca2+ activates PDH phosphatase
    • Inactive PDH is phosphorylated
      • ATP acts as a substrate for PDH kinase
    • Both phosphatase and kinase are part of PDC
  • Byproduct inhibition
    • Acetyl-CoA and NADH+H+ inhibits PDH
  • Acceptor control
    • Adequate amounts of NAD+ and CoA must be available for reaction to proceed.

Thiamine

  • Also known as Vitamin B1
  • Makes up a thiamine pyrophosphate (TPP)
    • Structurally, it’s a pyrimidine attached to a methyl, a thiozole, and 2 phosphates
    • Thiozole contains the carbon that accepts the acetyl group
  • Essential for metabolizing glucose
    • Co-enzyme for the decarboxylation of α-keto acids such as pyruvic acid and α-keto glutaric acid
    • Also a cofactor for transketolase
  • Pyruvate is converted to Acetyl-CoA by oxidative decarboxylation
  • α-ketoglutarate is converted to succinyl-CoA by oxidative decarboxylation
  • Nutritional sources: pork, whole-grain cereals, legumes, and enriched grain productions
  • Storage is limited as liver stores can be depleted in 12-14 days

Thiamine Deficiency

  • Deficiency of Thiamine can lead to disturbances in carbohydrate metabolism
    • Decreased transketolase activity, particularly in erythrocytes and leukocytes
    • Lead to cardiovascular and neurologic lesions and emotional disturbances
  • Beri-Beri
    • Develop neuropathy, fatigue
    • “Dry” and “Wet” types; “Wet” Beri-Beri is more severe
  • Wernicke-Korsakoff Syndrome
  • Can result from alcoholism-related nutrient deficiency

Lipoic Acid

  • Also called Thioctic acid
  • Essential component in metabolism
  • Not a dietary requirement
  • Co-factor is hydrogen-transfer reactions
  • Key role in oxidative decarboxylation of pyruvate to Acetyl-CoA and α-ketoglutarate to succinyl-CoA
  • Carboxyl group usually bound to an enzyme by an amide bond

Co-enzyme A

  • Universal carrier of acyl groups
  • Forms high-energy, activated thioesters
    • High acetyl transfer potential because of hydrolysis of thioester is more thermodynamically favorable than oxygen ester.
    • ΔG°’ = -7.5 kcal/mol
  • Contains a Pantetheine attached to a 3’Pi-ADP
    • Pantetheine is made up by β-mercaptoethylamine and pantothenate
    • β-mercaptoethylamine contains the reactive –SH group
    • Pantothenate or pantothenic acid is one of the Vitamin B’s
      • Cannot be synthesized and must be acquired in diet
      • No known human disease due to deficiencies

Coenzyme NAD+

  • NAD+ can be reversibly reduced to the NADH+H+ form
  • NAD+ consist of a nicotinamide component bound to a ribose phosphate and an AMP component
  • The nicotinamide carbon #4 is the carbon accepting the hydride H- ion when it is reduced
    • Nicotinamide is an amine derivative of niacin or nicotinic acid
    • Deficiencies in niacin cause pellagra
      • Symptoms: dermatitis, diarrhea, and dementia
  • A related particle called NADP+ also contains nicotinamide and, like NAD+, is a co-enzyme of many oxidoreductases
    • NAD+ participates in many catabolic reactions
    • NADP+ participates in biosynthesis reactions

Function of NAD+

  • A Hydrogen acceptor in oxidation of HC=OH to C=O
    • Hydrogen from same carbon
  • Oxidation via oxidative phosphorylation yields 2.5 ATP

Coenzyme FAD

  • FAD or flavin adenine dinucleotide is a co-enzyme of most flavoproteins.
    • Tightly bound to enzyme
    • Contains Vitamin B2 or Riboflavin
      • No major human disease associated with riboflavin deficiency
      • Component of prosthetic groups FMN and FAD of flavoprotiens
  • Consists of an AMP component attached to a C5 ribitol and a isolloxazine ring components
    • Reduction site is the #1 and #5 nitrogen of the isoalloxazine
  • FMN or Flavin mononucleotide is a 5’-phosphate derivative of riboflavin
    • FAD = FMN+AMP

Function of FAD

  • FAD is generally a hydrogen acceptor in forming C=C bonds
    • Hydrogens from adjacent carbons
  • Oxidation via oxidative phosphorylation yields 1.5 ATP

Anaplerotic Reactions

  • Intermediates such as oxaloacetate are required to keep the TCA cycle going.
  • Pyruvate + CO2 + ATP --> Oxaloacetate + ADP + Pi
    • Catalyzed by pyruvate carboxylase with biotin as a co-enzyme
      • Activated by Acetyl-CoA
    • Occurs when TCA cycle is deficient in oxaloacetate

Amphibolic Nature of TCA Cycle

  • Glutamate + Pyruvate <--> α-ketoglutarate + alanine
    • Catalyzed by transaminases
  • Aspartate + Pyruvate <--> oxaloacetate + alanine
    • Catalyzed by transaminases
  • Citrate can be converted to Acetyl-CoA
    • Citrate can be transported out of mitochondria where it is converted to Acetyl-CoA and Oxaloacetate by ATP-citrate lysase
      • Acetyl-CoA can be further converted to fatty acids
  • Succinyl-CoA can be converted to heme synthesis

Metabolism of Amino Acids

  • Methionine, serine, cysteine, threonine, and glycine can be converted to pyruvate
  • Tryptophan can be converted to alanine which can be converted to pyruvate
    • Transaminease can remove the –NH2 from alanine to convert o pyruvate
  • Tyrosine and phenylalanine can be converted to fumarate
  • Isoleucine. Methionine, and valine can be converted to succinyl-CoA
  • Histidine, proline, hydroxyproline, and arginine can be converted to glutamic acid
    • Glutamic acid can be converted to α-ketoglutarate

Other Reactions

  • Oxaloacetate can be changed to P-Enol-Pyruvate by P-Enol Pyruvate carboxykinase
    • P-Enol-Pyruvate can be converted back to glucose
  • Propionate can be converted to succinyl CoA
  • Pyruvate can be converted to acetyl CoA

Regulation of TCA Cycle

  • Rate of Acetyl CoA formation from pyruvate is regulated by increasing Ca2+, ATP, NADH+H+, and Acetyl CoA
  • Isocitrate dehydrogenase catalyzes the committed step in Reaction 3
    • Reaction requires ADP as a positive or stimulatory allosteric modulator
  • Reactions 3 and 4 are inhibited allosterically by ATP and NADH+H+ and stimulated by increased Ca2+
  • In general, the TCA cycle is regulated by the need for ATP, the presence of O2, NAD+, FAD, and presence of TCA cycle intermediates such as oxaloacetate